Project Summary/Abstract: HPV-associated cancer incidence is significantly elevated in cervical and at othersitesinHIV+patients.HIV+patientsacquiremorefrequentmulti-typeinfections,includingmanygenotypes infrequentlyseeninhealthyindividuals,andnottargetedbythecurrentHPVpreventivevaccines.Wepreviously developedacandidatetherapeuticandpreventiveHPVvaccine,pNGVL4a-CRTE6E7L2(CRTE6E7L2),which comprisesaDNAvectorencodingtheheatshockproteincalreticulinfusedgeneticallywithHPV16E6andE7 (that are obligately expressed in HPV malignancies) as well as the L2 capsid protein (a broadly protective antigen). We showed that fusion with calreticulin (CRT) profoundly enhances the potency of DNA vaccines in generating HPV antigen-specific CD8+ T cell mediated immune responses even in CD4-depleted animals. In addition, vaccination with the CRTE6E7L2 DNA vaccine induces both L2-specific neutralizing antibodies and protectionfromexperimentalvaginalchallenge.ThesefeaturesmaketheCRTE6E7L2DNAvaccineparticularly promisingforuseinHIV+patients,achallenginggrouptotreat,andtopreventmultipletypesofHPVinfections. Although DNA vaccines are relatively safe and well suited for multiple administrations, they generally exhibit suboptimal immunogenicity when administered by conventional intramuscular needle injection, likely reflect inefficienthostcelltransduction.WehavepreviouslyshownthatelectroporationisamuchmoreeffectiveDNA vaccine administration method to generate HPV-specific CD8+ T cell immune responses as compared to conventionalintramuscularinjectionorepidermaldeliveryviagenegun.Thus,thegoalofthisproposalistouse the Ichor TriGrid? Delivery System Electroporation Device, which has been used in multiple clinical trials, for intramuscularadministrationoftheCRTE6E7L2DNAvaccineatescalatingdosesinbothHIV?andHIV+patients with HPV16-associated high-grade cervical intraepithelial neoplasia grades 2 and 3 (CIN2/3), and to examine thesafety,virologic,anddiseaseoutcomes.TheSpecificAimsofthisstudyareto(1)evaluatethesafetyand toxicity of CRTE6E7L2 administered via electroporation in HIV? and HIV+ patients with HPV16+ CIN2/3;? (2) characterizetheHPV16E6/E7-specificcell-mediatedandhumoralimmuneresponsesinHIV?andHIV+patients with HPV16+ CIN2/3 vaccinated with CRTE6E7L2 via electroporation;? (3) characterize L2-specific humoral immune responses in HIV? and HIV+ patients with HPV16-associated CIN2/3 upon vaccination with CRTE6E7L2DNAvaccineviaelectroporation;?and(4)determinetheHPVloadandhistopathologicalchanges inthelesionanditsmicroenvironmentinHIV?andHIV+patientswithHPV16-associatedCIN2/3upontreatment withCRTE6E7L2DNAvaccineviaelectroporation.Thesuccessfulimplementationofourproposalwillprovide a new methodology for the treatment of HPV-associated high-grade squamous intraepithelial lesions in HIV? andHIV+patients.